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Annals of Botany 97: 11–27, 2006
doi:10.1093/aob/mcj001, available online at www.aob.oxfordjournals.org
INVITED REVIEW
Epigenetics and its Implications for Plant Biology 2. The ‘Epigenetic
Epiphany’: Epigenetics, Evolution and Beyond
R . T . G R A N T - D O W N T O N * and H . G . D I C K I N S O N
Department of Plant Sciences, University of Oxford, Rodney Porter Building, South Parks Road,
Oxford OX1 3RB, UK
Received: 6 March 2005 Returned for revision: 23 May 2005 Accepted: 22 August 2005 Published electronically: 31 October 2005
Scope In the second part of a two-part review, the ubiquity and universality of epigenetic systems is emphasized,
and attention is drawn to the key roles they play, ranging from transducing environmental signals to altering gene
expression, genomic architecture and defence.
Key Issues The importance of transience versus heritability in epigenetic marks is examined, as are the potential for
stable epigenetic marks to contribute to plant evolution, and the mechanisms generating novel epigenetic variation,
such as stress and interspecific hybridization.
Future Prospects It is suggested that the ramifications of epigenetics in plant biology are immense, yet unappreciated. In contrast to the ease with which the DNA sequence can be studied, studying the complex patterns
inherent in epigenetics poses many problems. Greater knowledge of patterns of epigenetic variation may be
informative in taxonomy and systematics, as well as population biology and conservation.
Key words: Epigenetics, ploidy, hybrids, DNA methylation, histones, RNA, chromatin, silencing, transgenes, transposons,
genome evolution, plant population biology, plant systematics, variation, heritability, plant development.
INTRODUCTION
As will be obvious from the first part of this review (GrantDownton and Dickinson, 2005), it is now evident that epigenetic systems are essential for genomic function. In terms
of genomic structure and its integrity, epigenetic systems
appear to be all-controlling. The formation and maintenance
of the major structural units of the chromosome defined
by their specialized heterochromatic state—the centromeric
structures and telomeric structures—appears to be determined by epigenetic systems. Direct evidence for centromeric heterochromatin in plants being determined by
transcription of repetitive DNA structures is now accumulating. Transcripts from these repetitive sequences are able
to form aberrant RNAs and dsRNA which are then
processed to siRNAs and act to direct and maintain
sequence-specific heterochromatin formation. The resulting
siRNAs appear to be rare and only recently have they been
detected directly. It would seem that RNA-based systems
are the singular most important mechanism for generating
heterochromatin of all types, whether constitutive structural
chromatin or heterochromatin directed to other sequences.
The RNA-based processing of aberrant transcripts also acts
as a surveillance system for unsilenced transposon
sequences in the genome, with inactivation of any active
elements through DNA methylation and heterochromatin
formation. These elements can be deleterious to the genome
through their mutagenic effects during movement and their
unregulated movement would be damaging over a longer
period of time. Already, the activation of such elements in
backgrounds such as ddm1 has been shown to produce novel
transposon-induced mutants. Naturally, the RNA system for
* For correspondence. E-mail [email protected]
detection of dsRNA viral transcripts is essential to the plant
for protection against these pathogens, with its direct trigger
of invading RNA virus destruction. Therefore the dual roles
of structuring the genome and its defence are united in
epigenetic pathways (Waterhouse et al., 2001).
The flip side of control over silencing, i.e. control over
gene expression in euchromatic regions, is naturally the
domain of epigenetic systems. Structural features of chromatin, from higher-order architecture to positioning of the
nucleosome on a specific sequence of a promoter, dictate
whether transcriptional machinery such as transcription
factors can bind and effect transcription from a sequence.
Chromatin states act in conjunction with transcription factors to regulate which genes are transcribed, and to what
extent they are transcribed. Even if a sequence is transcribed
and transcripts are abundant, post-transcriptional regulation
can occur via the miRNA-induced degradation system.
What seems the most important factor about these epigenetic systems of control is that the changes are dynamic and
typically can be rapidly induced in response to stimuli.
Epigenetic systems therefore act as the conduit for information, both developmental and environmental in origin, to
initiate short- and long-term gene expression changes. In
plants, tracing the pathway from initiating signal to gene
expression change via epigenetic systems is rather lagging
behind the same kind of work in eukaryotic model systems
such as yeast. In terms of developmentally regulated gene
expression changes, the most detailed example is the
expression of the phaseolin gene in French bean. This
gene encodes a major seed storage protein and its expression
is under very tight developmental regulation, restricted to
the later phase of embryogenesis. A full review of this
model system for developmental regulation of a gene via
chromatin changes can be found in Li et al. (2001).
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Grant-Downton and Dickinson — Epigenetics, Evolution and Beyond
EPIGENETICS AS THE CONDUIT FOR
ENVIRONMENTAL SIGNALS
Sensing environmental changes and initiating a gene
expression response is most important for plants as sessile
autotrophs. Responses can be both short term (e.g. synthesis of protectant molecules against stress) and long
term (e.g. phenotypic plasticity, with alteration of developmental programmes) because plant tissues typically have
the benefit of no rigid or set developmental plan. Epigenetic
systems must be part of the relay from sensing a change in
the environment to a change in gene expression. Their ability to alter rapidly and reversibly, yet with the potential to
keep a stable ‘memory’ through many cell divisions, must
be a key to the flexibility of plant responses to the environment. Perhaps this is why plant epigenetic systems are complex and contain some unique constituents not seen in other
eukaryotes. Surprisingly, not much is known about epigenetics and plant responses to environmental change, with
one major exception. This exception is the regulation of
flowering time (i.e. initiating inflorescence development)
in Arabidopsis thaliana in response to long periods of
cold temperatures, known as vernalization. Some genotypes
of this annual species require an extended period of cold
temperatures before commitment to flowering, behaving as
‘winter annuals’, with this vernalization requirement preventing flowering until favourable spring weather arrives.
Other genotypes do not require cold treatment to induce
flowering. The major genetic differences between these
genotypes have been identified to two loci, FRIGIDA
(FRI) and FLOWERING LOCUS C (FLC). Molecular studies have shown that FLC acts as a repressor of flowering,
whilst FRI acts to boost the levels of FLC, so these two
proteins act synergistically to block flowering. How does
exposure to cold block this repression of flowering? Exposure to cold must be ‘recorded and stored’ by the plant, as
when temperatures increase and mitotic cell divisions continue, the memory of exposure to cold is still retained as
is the competence to flower. Storing this as epigenetic
information has now been shown to be the key to the vernalization response. With exposure to cold over a significant
length of time, the chromatin at the FLC locus changes, with
reduction of acetylation at H3 K9 and K14 sites, and
increase in methylation of the H3 K9 and K27 sites, corresponding to the formation of heterochromatin (Bastow
et al., 2004; Sung and Amasino, 2004). Furthermore, it
has been reported that this locus shows reduced H3 K4
trimethylation, a mark that has been associated with transcriptional activation, after vernalization (Amasino, 2004).
It appears that the ultimate outcome of lengthy cold exposure is formation of heterochromatin and silencing of FLC,
repressing the main repressor of flowering. Interestingly,
FLC alleles with transposable elements inserted in an intron
are down-regulated and flower rapidly without vernalization
as its presence induces stable heterochromatin formation
at the locus, mimicking vernalization-induced heterochromatin (Amasino, 2004). Mutants that block responsiveness
to cold have also been identified. Two of the mutants
correspond to chromatin-remodelling proteins, with
VERNALIZATION2 (VRN2) a Pc-G protein (Bastow et al.,
2004) and VERNALIZATION INDEPENDENCE3 (VIP3),
forming a repeat-rich protein which is likely to be involved
in forming the scaffold for assembling chromatin-remodelling protein complexes (Zhang et al., 2003), fitting with
epigenetic pathways regulating vernalization responses.
The most significant discovery is that mutations in three
Arabidopsis genes homologous to components of the yeast
PAF1 complex, a complex which recruits a histone methyltransferase (SET1) catalysing H3K4 trimethylation, all promote vernalization-independent early flowering, block FLC
expression and reduce FLC H3K4 trimethylation (Zhang
et al., 2002; He et al., 2004; He and Amasino, 2005). Moreover, altered flowering time has been regularly seen as a
pleiotropic effect of other mutants in the epigenetic machinery. What is not known is any of the molecular detail of how
the cold is sensed over a continuous lengthy period and the
signal relayed to effect heterochromatization of FLC. By
some mechanism, a long duration of cold is measured and
committed to ‘epigenetic memory’, as short durations of
cold are insufficient to acquire competence to flower;
Amasino (2004) has speculated that this cold sensing
may be via titration effects of antagonistic enzymes in a
signalling pathway where one enzyme has different
temperature-response kinetics. Clearly there is still much
work to be done in revealing the full working detail of the
pathway from environmental stimulus to gene expression
change even in this model system.
EPIGENETIC HERITABILITY IN PLANTS:
LAMARCK’S LAST LAUGH?
Epigenetic states and their heritability in plants
How the plasticity of plant growth and development may
be a consequence of sophisticated epigenetic systems that
allow sensitive and rapid gene expression changes to be
effected has been discussed above. Another interesting consequence of plant developmental programmes is that there
is no dedicated germline set up, segregated and maintained
from early development onwards. Instead, cells giving rise
to the germline develop de novo from the somatic tissues.
This allows the opportunity for any stable epigenetic
information acquired by the chromatin–DNA structures of
the somatic tissues to be transmitted to the next generation,
provided no epigenetic resetting system that deletes such
acquired information is active. Clearly, the structurally
important epigenetic information of the centromeres and
telomeres is not deleted in this way during reproduction
but this is just the start. How in paramutation epigenetic
states at some loci can be heritably transmitted and influence
susceptible (paramutable) alleles in the next generation, has
been discussed in previous paragraphs. Spontaneous
epialleles, such as those identified at SUPERMAN and
Lcycloidea loci, and epialleles at many other loci induced
by events such as interspecific hybridization and a background with compromised epigenetic machinery, have been
shown to be heritable with considerable fidelity. Silent
(heterochromatic) transposable elements and other repetitive sequences (such as transgene structures) located
in euchromatic regions also retain their state with great
Grant-Downton and Dickinson — Epigenetics, Evolution and Beyond
stability from generation to generation. This could be
explained by either stable transmission of epigenetic
marks from generation to generation, or a short phase in
the meiotic-gametophytic development in which there is an
altered epigenetic state (e.g. release of silencing by loss of
heterochromatization) followed by rapid and consistent reestablishment. Evidence for both scenarios exists. Perhaps
the most interesting evidence comes from analysis of Arabidopsis met1 mutants in the male gametophyte. Heterozygotes for the mutation have been shown to accrue changes to
DNA methylation in first-generation offspring, even though
the mutation behaves recessively (Saze et al., 2003). This is
because after meiosis the male germ line undergoes further
mitotic divisions; those meiocytes lacking functional MET1
undergo cell division without the maintenance methyltransferase to preserve DNA methylation with absolute fidelity,
leading to sperm cells with aberrant methylation patterns.
By uncovering evidence for an essential role for MET1 in
keeping epigenetic order through gametophytic development, this work implies that DNA methylation could be the
major repository of epigenetic information in plants from
generation to generation, as at this phase of the life cycle no
‘back-up’ of chromatin information (such as histone marks)
exists. This discovery implies at least some chromatin therefore has to be set up and reprogrammed de novo from the
DNA sequence and methylation marks with the next generation. This is fitting with observations of gross, global
changes in chromatin during gametophytic development
in plants, though detail of this gametophytic reprogramming
is scarcely understood and appears to vary considerably
from species to species. Interestingly, work in transgene
silencing systems has revealed that the methylation in the
coding region in PTGS silenced lines is not persistent from
generation to generation in the absence of the trigger,
whereas in TGS silenced lines there is evidence for some
persistence of silencing and retention of symmetrical DNA
methylation even after the inducing construct has been segregated away (Aufsatz et al., 2002). It is important to point
out that the naturally occurring hypermethylated Lcyc allele
in Linaria vulgaris shows quite stable inheritance of DNA
methylation in both the promoter and coding regions (Cubas
et al., 1999) and the same is true of SUPERMAN epialleles
(Kishimoto et al., 2001). However, the exact mechanism of
their spontaneous genesis is not known, though conventional transposon insertion events have been ruled out, making these ‘exceptions’ awkward. Further consideration of
heritable phenomena will be found in later discussion of
epigenetic phenomena in hybridization. This evidence does
suggest that some acquired information sealed in the epigenetic state can be passed unperturbed from parent to
offspring.
Epigenetic rewriting and the gametophyte
Equally, though, there is evidence in other plant taxa for
global changes in DNA methylation in development of the
gametophyte. Oakeley et al. (1997) report the global reduction in DNA methylation of the generative nucleus compared with the vegetative nucleus in Nicotiana pollen
development after using immunocytological detection.
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An updated study using antibodies raised to the different
histone marks would be hugely informative. Could these
changes allow a slight release of the silenced epigenetic
state at many loci, with the ‘sensing’ mechanisms that detect
‘aberrant’ transcripts resetting constitutively heterochromatic regions anew for the next generation? Wholesale
release of silencing of transposons and repetitive sequences
across the genome certainly seems unlikely to take place
for two reasons. First, it seems a remote prospect that the
epigenetic pathways could fully cope with a deluge of previously suppressed transcripts and, secondly, the mutagenic
nature of activated transposable elements would be deleterious, particularly in haploid gametophytes. Indeed, it is
tempting to speculate that the presence of specialized gametophytic chromatin states may act to allow partial release
of transcription, with a limited increase in transcription
from otherwise heterochromatic regions but without an
overwhelming derepression of transcription. Using
sequences such as transposable elements, centromeric
repeats and silenced transgenes to determine their level
of transcription and the levels of derived siRNAs in the
stages and different cells of gametophytic development
would be required to ascertain whether subtle changes in
transcriptional potential across the genome are initiated. It is
fitting to note that transcriptome analysis of maize sperm
cells has revealed that a substantial proportion of sequences
identified are transposons, with a published estimate of 8 %
being retrotransposons (Engel et al., 2003) and an updated
estimate of 19 % (McCormick laboratory website, http://
www.pgec.usda.gov/McCormick/McCormick/mclab.html).
Epigenetic memory and environmental responses
The subtle nuances of the heritability of epigenetic states
have yet to receive enough attention, although it certainly is
not an outright Lamarckian mechanism, even in plants. The
work on vernalization is a clear example of plants acquiring
and utilizing information from the environment without the
epigenetic information becoming heritably stable. Vernalized plants show propagation of the epigenetic memory of
cold through mitosis but not meiosis—offspring from coldexposed plants do not show altered flowering times. A system must exist in reproductive development that re-sets this
specific heterochromatic state after the signal to initiate
flowering has been conveyed. Indeed, there appears to be
no known stable epimutations/epialleles for FLC that affect
flowering time, only sequence variants such as transposon
insertions. Why is this particular heterochromatic state reset so efficiently? One reason may be that the FLC locus is
part of a domain dedicated to vernalization and it may be
distinguished by its own unique chromatin markings. Recent
work has shown that FLC and a neighbouring locus (of
unknown function) are co-ordinately affected by vernalization treatments (Finnegan et al., 2004). Moreover, transgenes inserted into this region acquire vernalization
responsiveness and, when the sequence of this region itself
is moved to other parts of the genome, it retains its vernalization responsiveness and can affect the neighbouring
sequence. Collectively, this points to the sequence of
the FLC region as the key to acquiring a particular
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Grant-Downton and Dickinson — Epigenetics, Evolution and Beyond
heterochromatin information state, which can then spread
outwards to the neighbouring sequence. Further dissection
of the properties of this domain to explore why and how it
subsequently loses this heterochromatic mark would be
most valuable.
To what extent is the FLC domain representative of how
the plant genome is constructed? It has been argued that
other such transcriptional domains are common throughout
the genomes of plants (Finnegan, 2001). If each has autonomous mechanisms during the reproductive development
for eliminating or resetting epigenetic information acquired
during growth, this would limit the amount of epigenetic
information passed from generation to generation. However, the apparent rarity of transgene insertions acquiring
novel expression profiles from the surrounding sequence
(Schubert et al., 2004), and evidence from epigenetics in
hybrid systems (discussed below), leaves this subject to
debate. The most pressing question is—how many DNA
sequences can show metastable epigenetic behaviour,
with the potential to occupy more than one epigenetic
state (i.e. form epialleles or epimutations) that are heritable
and affecting gene expression? As yet, it is only possible to
infer answers, but with developments in analysis of transcriptional profiling and epigenetics (such as bisulfite
sequencing and CHiP), more information is likely to accrue.
Even if a fraction of the genes in plants have the potential to
form sets of epialleles, it would be a tremendously important, yet hitherto undetected, source of variation. Even in
mammals there are examples of epialleles, such as at
the axin and agouti loci, where the normal resetting of
the epigenetic state during reproduction is escaped which
allows trans-generational inheritance of epigenetic states
with its consequences for expression patterns of the gene
and subsequent phenotype (Rakyan et al., 2003; Chong and
Whitelaw, 2004). If, as seems logical, the capacity to form
epialleles is determined to some extent by DNA sequence,
this raises new questions about the interplay between
sequence evolution and epigenetic evolution.
EVOLUTION AND EPIGENETICS
IN PLANTS
Epigenetics of plant genome evolution
In hindsight, it now seems strange that plant biologists have,
for many years, discussed evolution of plant genomes and
plant populations whilst only considering the DNA and
protein sequence as the determinants of phenotype. Prior
to the extensive molecular exploration of epigenetics,
early ideas on epigenetics and evolution were reviewed
and discussed by Jablonka and Lamb (1989). It is now an
exciting time for plant biologists interested in evolution as
the new data on epigenetic systems in plants can be assimilated into a more complete understanding of the field. In this
section it is considered how essential epigenetics have
become in understanding various evolutionary problems
and where following this avenue to its logical conclusion
may eventually lead.
What drives the formation of diversity in the genome?
At the level of the DNA sequence, alterations such as
deletions, point mutations and chromosomal rearrangements have been the main focus for decades and the prevailing consideration in how genomes and populations
evolve over time. There is no dispute that such alterations
are essential to generating variation, both ‘neutral’ and
‘non-neutral’ upon which selection can act directly. Interestingly, there is evidence that the mutational frequency of
methyl-cytosine is substantially greater than unmodified
cytosines (e.g. Rideout et al., 1990). The mutations are
formed from the deamination of methyl-cytosine to thymine. This implies that there is a mutational penalty for
marking sequences by methylation, which would be more
severe for densely methylated sequences such as repetitive
DNA. It is tempting to speculate that patterns of DNA
methylation can significantly bias the mutational frequency
of the marked sequence in plants. Transposons and repetitive
DNA targeted for RdDM and heterochromatization may
have higher rates of mutation than euchromatic regions,
perhaps aiding in defence. This mechanism has been proposed to operate in elimination of ectopic promoters in the
coding regions of genes in plants (Tran et al., 2005).
Transposons and epigenetics in genome evolution
This brings us to the next major source of variation in the
plant genome—transposon-induced variation. Since the discovery of these selfish mobile elements, their modifying
effects on the host genome have become well documented
and will be but briefly mentioned here (see Kidwell and
Lisch, 1997, 2001; Dimitri and Junakovic, 1999). The first
notions of their importance in shaping genomes came from
McClintock (see McClintock, 1984). Their effects range
from the outright mutagenic, e.g. disruption of coding
genes by insertion of a mobile element, the formation of
‘footprints’ in insertion and excision cycles, to more subtle.
For instance, ectopic recombination can occur between
homologous elements in meiosis, leading to chromosomal
rearrangements, and it is possible that ‘macrotransposons’
can form, capable of shifting even relatively large sequences
of trapped host DNA to new sites in the genome (Gray,
2000). Clearly, most outcomes of transpositional activity in
an evolved, structured genome will be neutral to deleterious,
making the defence system described above essential to
prevent dangerously high levels of transposition. Of course,
the defence system that induces heterochromatin formation
of detected transposon sequences may actually cause some
of the deleterious effects itself, by silencing neighbouring
host sequences. In recent years, there has been a considerable shift in the appreciation of these mobile elements and
the view of them as selfish parasites has become mollified to
treating them as genomic symbionts. Many plant genomes
are enormously rich in these kinds of sequences yet efficient
mechanisms to rid genomes of such sequences have evolved
in other eukaryotes, suggesting that their co-habitation has
some benefits. Nowhere is this more evident than in the
structural centromeric and telomeric regions of genomes
that are essential for genomic integrity and rich in such
sequences. Integration of transposable elements within or
close to coding genes has been demonstrated to generate
novel phenotypic variation in plants (Kumar and Bennetzen,
Grant-Downton and Dickinson — Epigenetics, Evolution and Beyond
1999) with examples from cultivated plants such as maize
(Wessler, 1988) and Antirrhinum (Coen et al., 1986) known
for many years. However, there is a considerable gulf
between populations of cultivated plants and plants in populations where natural selection hones the genome. A boost to
the view that transposable elements can shape the genome
and gene expression patterns in nature has been the finding
that ‘fossil remnants’ of transposable element sequences are
ubiquitous in euchromatic gene sequences. Statistical surveys (e.g. Bureau et al., 1996) have found that many plant
genes have close associations with transposable elements.
White et al. (1994) have identified plant genes that probably
have coding regions of genes adopted from copia retrotransposon gene sequences. The same study revealed that many
genes have degenerate transposable element sequences in
their upstream and downstream flanking sequences; in some
cases, this sequence is known to have a direct regulatory
effect on the expression of the gene. Perhaps the most interesting and complete example is the imprinted FWA gene,
shown to be epigenetically sensitive, which has been shown
to be intimately associated with SINE retrotransposon
sequences (Lippman et al., 2004). siRNAs corresponding
to this sequence were also found in this study and the data
points to FWA imprinting and transcription being directly
controlled by this retrotransposon sequence. Coding genes
may ‘adopt’ integrated or nearby transposable element
sequences on a frequent basis, making such elements a
major force for generating variation and novelty in the
genome; indeed, Kumar and Bennetzen (1999) speculate
that perhaps all plant genes will be shown to contain a
transposable element legacy. As epigenetic systems are
the major regulators of these elements, it is a case for
‘guilt by association’ in their contribution to the molecular
and phenotypic evolution of plants.
Another connection between repetitive DNA and the
evolution of epigenetic control of gene expression has
recently been revealed. By analysing the sequences of
microRNA genes in Arabidopsis, it has been shown that
these genes are likely to arise from inverted repeats of
coding genes. Allen et al. (2004) have identified miRNA
genes from Arabidopsis that appear to be intermediate
between inverted repeats and typical miRNA genes, as
their sequence shows considerable homology to the target
gene outside the region of binding of the processed miRNA.
Indeed, one of these ‘intermediate’ genes, ASRP1729, did
not seem to have behave as an miRNA, as it did not have a
targeting function and was insensitive to DCL-1 mutations
for its processing, possibly representing an intermediate
stage before miRNA function has evolved. It seems plausible that these ‘intermediates’ may become trans-acting
siRNAs (Peragine et al., 2004; Vazquez et al., 2004). In this
manner, new miRNA genes controlling novel expression
patterns of the target genes could arise without much difficulty. Voinnet (2004) commenting on this work suggests
that, ‘given their highly evolving nature, young small RNAs
are ideally suited to convey rapid adaptation and will probably be more abundant in plants subjected to stress’. In support of this idea, it is interesting to note that a strong
candidate for a non-coding RNA involved in transcriptional
stress responses was identified by T-DNA tagging in the
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resurrection plant Craterostigma plantagineum (Furini et al.,
1997; Bartels and Salamini, 2001). This sequence is involved
in negatively regulating desiccation-tolerance genes and its
sequence shows closest similarity to SINE-like retrotransposons. No homologues have been detected in Arabidopsis;
it would be interesting to determine how quickly this
regulatory RNA evolved by examining whether similar
sequences exist in closer relatives in the Scrophulariaceae
and Gesneriaceae. At the other extreme, there is evidence
for astonishing conservation of miRNAs and their target
sequences, indicating that once this regulatory system is
in place and controlling transcript abundance it can become
highly canalized. The mRNAs of class-III HD-Zip transcription factors from all lineages of land plants have retained
an miR165/166 binding site, and evidence for cleavage at
this site from several species was also forthcoming (Floyd
and Bowman, 2004). The retention of miRNA regulation of
these genes may span more than 400 million years. However,
there is no evidence for overlap between miRNAs and genes
targeted by miRNAs in plants and animals.
Evolution through mutations in genes
governing epigenetic systems
Another means by which epigenetic systems could generate novel variation in plant populations is through mutations in genes of major regulatory effect, e.g. genes like
MET1 and DDM1. As discussed above, loss of function does
not necessarily prove lethal to the plant and their absence
can lead to the generation of stable epialleles of genes of
developmental and morphological importance, as well as
activation of transposable elements. Importantly, there is
evidence that some epialleles can be maintained independent of the loss-of-function mutation that provided their
genesis; restoration of epigenetic function by crossing
back to wild type may rescue epigenetic regulation systems
but with the retention of the epiallele. The loss of methylation of the FWA locus in Arabidopsis (Soppe et al., 2000)
is heritable and induces ectopic expression of the FWA
protein which results in a late-flowering phenotype. In
fact, FWA is not expressed at all in adult tissues of the
wild type, but instead is restricted to the central cell of
the megagametophyte and the resulting endosperm tissue
after fertilization (Kinoshita et al., 2004), where expression
is correlated with reduced methylation derived from the
DNA glycosylase activity of DEMETER in the central
cell. The default state of FWA is methylated and silenced,
except in terminally differentiated reproductive tissues, but
its reactivation and ectopic expression by an indiscriminate
demethylation event results in an unrelated and rather unexpected late-flowering phenotype. The floral homeotic genes
SUPERMAN and AGAMOUS both show heritable hypermethylation and gene silencing, paradoxically in the
hypomethylated backgrounds of met1 and ddm1 mutants.
The dramatic phenotypes of their silencing are an excellent
illustration of epialleles.
A more enigmatic example is the generation of overexpressing bal epialleles of the CPR1 (constitutive expressor of pathogenesis related proteins 1) locus (located in the
chromosome 4 Resistance gene cluster) in ddm1 mutant
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backgrounds (Stokes and Richards, 2002; Stokes et al.,
2002). bal epimutants show phenotypes such as twisted
leaves, dwarfing and reduced fertility, similar to cpr1-1
from ethyl methyl sulphonate exposure. Surprisingly,
these appear to be two different epialleles and neither associated with nucleotide changes. Intriguingly, selfed offspring from bal/cpr1-1 heterozygotes produce approx.
20 % phenotypically wild-type plants, perhaps derived
from epigenetic destabilization and reversion mediated by
pairing interactions (Stokes and Richards, 2002). There is
also direct evidence that reactivation of transposable elements in the ddm1 background can produce new insertions
with phenotypic consequences (Miura et al., 2001). Indeed,
once activated by ddm1, transposons can retain their activity
even when functional DDM1 is restored (Lippman et al.,
2004). It does not seem too dramatic to suggest that saltational changes in the heritable epigenetic marks and phenotype could arise from even short-term exposure to defective
activity of one or a few genes in the epigenetic system.
Stress-induced epigenetic change
Environmental and biotic stress may also induce the
formation of novel epialleles. Mild inhibition of chaperone
activity of the Hsp90 protein in plants by specific pharmacological agents has been shown to produce a diversity of
phenotypic changes in Arabidopsis seedlings, such as alterations to plant size, leaf shape and pigmentation (Quietsch
et al., 2002). This may be due to the fact that the chaperone
folding activity of Hsp90 can partially counteract the effect
of mutations in the DNA sequence that generate amino acid
changes in proteins, rendering them ‘neutral’ in effect on the
protein function. When Hsp90 efficiency is reduced, accumulated variation of this kind is rendered non-neutral and
alterations to protein function ensue, exposing them to
selection (Sangster and Queitsch, 2005). Definitive genetic
proof that the phenotypic effects seen in Arabidopsis are
derived in this way has not been presented. Another plausible scenario is that inhibition of full Hsp90 activity can
also decrease the functional efficiency of the epigenetic
machinery, e.g. decreasing the fidelity of epigenetic
marks such as cytosine methylation, that results in a heritable change to epigenetic and gene expression states.
Confirmation that this is the case from molecular epigenetic
studies is still lacking. It follows that any environmental
insult, such as extreme heat stress, could perturb the activity
of epigenetic regulation and heritable effects on epigenetic
marks and gene expression changes could arise.
Biotic stress in the form of pathogen attacks may also
generate epigenetic aberrations. There is evidence that the
Bs1 transposable element of maize has been reactivated by
viral infection (Johns et al., 1985). Viral infections may
bring about epigenetic changes of this ilk by the virtue of
proteins they encode to counteract the defensive RNA pathway machinery of the host plant. The role of these proteins
is to inhibit the activity of the defence pathway that destroys
viral transcripts, but it also appears that the other RNA
pathways are affected. Some of these viral suppressors of
silencing bind small RNAs and affect their processing,
whilst others such as P1/Hc-Pro may operate by inhibiting
the protein components of the pathway. For example, there
is evidence that the P1/HC-Pro suppressor of viral silencing
interferes with miRNA pathways in Arabidopsis where it
causes aberrant processing of some miRNAs and developmental aberrations (Kasschau et al., 2003); in tobacco
accumulation of siRNAs from transgenes and endogenous
miRNAs are differentially affected by P1/Hc-Pro (Mallory
et al., 2002). Dunoyer et al. (2004) expressed five different
viral silencing suppressors in Arabidopsis, all of which
blocked transgene PTGS, but only three (P1/Hc-Pro, P19
and P15) affected plant morphology. Of these three, P1/HcPro and P19 altered miRNA accumulation, whilst all three
prevented degradation of miRNA targets. It seems probable
that many of the gross abnormalities of plants infected with
viruses forming dsRNA intermediates may be due directly
to the upset of miRNA processing that regulates normal
development. It is just conceivable that, in a manner akin
to the formation of pseudogenes, viral transcripts may be
incorporated into the host genome and ‘adoption’ of a viral
gene encoding a silencing suppressor could bring about
dramatic, heritable phenotypic effects by affecting the regulation of miRNAs.
Is epigenetic change important in adaptation?
Evidence for the influence of single epimutations or epialleles on the stable evolution of plant characteristics
remains absent, perhaps because changes in DNA sequence
during any length of evolutionary time will obfuscate the
epigenetic contribution. Speculation is irresistible though.
Chandler and Stam (2004) have speculated that paramutation may be a mechanism for transmitting environmentally
adapted expression patterns. On a more specific level, it is
fascinating that the differences in floral symmetry and floral
organ number between Antirrhinum and the related
Mohavea is derived from altered CYCLOIDEA expression
(Hileman et al., 2003). The adaptive basis of this morphological change is likely to be pollination-based, with
Mohavea flowers mimicking the symmetrical flowers of
the unrelated Mentzelia in the same desert habitat. Could
epigenetic phenomena have initiated this dramatic shift
between two adaptive peaks? Epigenetic explanations
may be irresistible for other morphological changes that
have repeatedly occurred in plant evolution. The timing
of transition from the juvenile vegetative to adult vegetative
to reproductive development can differ considerably even
between closely related taxa. Screens for mutations in
Arabidopsis genes that alter the timing of these phase
changes have revealed several key genes confirmed to be
involved in epigenetic processing such as HASTY (Bollman
et al., 2003) and SDE1/RDR6 (Peragine et al., 2004). Not
only does this implicate a number of endogenous small RNA
species as essential for controlling the timing of key developmental phase transitions in the life of the plant (Peragine
et al., 2004), it also hints that mutations in these loci have the
potential to heritably alter how a plant proceeds through its
life cycle. For instance it may take just a few key mutations
to generate plants that proceed very rapidly through their life
cycle (as seen in many desert ephemerals) or retain persistently the features of juvenile vegetative growth.
Grant-Downton and Dickinson — Epigenetics, Evolution and Beyond
Perhaps the most likely situation where epigenetic
changes assist in the generation of an advantageous phenotype is the evolution of apomixis. The evolution of
apomixis, in particular autonomous apomixis where an
endosperm develops with no paternal genomic contribution,
has been seen as problematic to the parent–offspring conflict model (Haig and Westoby, 1991). As apomixis has
evolved repeatedly in the angiosperms, there must be pathways that allow its genesis. Experimental evidence suggests
that alterations in the epigenetic machinery that control
gametophyte development, imprinting and endosperm
development are the basis of endosperm development
without fertilization. Mutations in several Arabidopsis
genes [two of the Polycomb group, MEDEA (MEA) and
FERTILIZATION-INDEPENDENT SEED1 (FIE1), as
well as FERTILIZATION-INDEPENDENT ENDOSPERM1
(FIS2)] promote proliferation of the central cell prior to
fertilization (reviewed in Spielman et al., 2003). Yet none
of these mutants promote any substantial development or
differentiation of endosperm-like structures. However, loss
of function of MET1, leading to hypomethylation of the
maternal gametophyte in fie1 mutants, leads to the production of endosperm more closely resembling sexually derived
endosperm (Vinkenoog et al., 2000). In the absence of
fertilization, the spontaneous formation of endosperm-like
syncytial structures has also been reported from mutants in
the Arabidopsis MULTICOPY SUPPRESSOR OF IRA 1
(MSI1) gene (Köhler et al., 2003). The MSI1 protein is a
WD40 repeat protein that forms part of Polycomb group
complexes (discussed in Part 1). Recent work by Guitton
and Berger (2005) has confirmed that these endosperm-like
structures are derived from central cells, and report that
msi1 mutants also initiate the development of non-viable
but polarized, parthenogenetic embryos from the egg cell. It
seems conceivable that full-blown apomixis could evolve
from just a few mutations in genes regulating key epigenetic
processes.
EPIGENETICS OF PLANT
HYBRIDIZATION
Perhaps by virtue of the highly flexible development
allowed by their responsive epigenetic systems, plants
are particularly adept at hybridization. Intriguingly, the ability to hybridize does seem to have a taxonomic bias with
some families showing far greater rates of hybrid formation
than others (Ellstrand et al., 1996). What does seem common in hybridization events in plants is that it can unlock
variation not seen in either parental species. This has been
exploited in cultivated plants in particular, but there is also
evidence that hybridization could be of major importance in
the evolution and diversification of plants in nature (for
general reviews of plant hybridization see, Stebbins,
1950; Grant, 1971). Rather than being ‘dead-ends’, fertile
plant hybrids may make an important contribution to the
evolution of plant populations in many taxa, though the
extent of their importance remains subject to debate.
Where does all the novel variation come from in hybrids?
This has been attributed in the past to the effect of
17
combining the genomes and proteomes of two previously
separate, distinct entities; divergence in DNA and protein
sequences would allow novel combinations in hybrids with
the outcome being altered phenotypes with characteristics
different to either parent. In fertile hybrid populations,
‘transgressive segregation’ has become a popular hypothesis to explain the generation of characteristics that are
extreme, in both positive and negative directions, compared
with the parents (Rieseberg et al., 1999, 2003). The genetic
model that explains the generation of ‘extreme’ phenotypes
is the segregation of distinct alleles, derived from the genetically divergent parents, of different quantitative effects
on a characteristic, in hybrid populations. The combinations
of these quantitative trait loci (QTL) alleles from both parents that have additive effects in the same direction generate
characteristics in excess of either parent. These combinations, of course, are not possible in the isolated parental
populations and only become possible in the hybrids.
The Arabidopsis hybrid model system in epigenetic studies
However, investigation of the epigenetics of hybrids, in
particular the use of allopolyploid hybrids between
Arabidopsis thaliana and A. arenosa as a convenient model
system, has been a revelation. This model system is useful
as A. thaliana is already the major model plant and the
hybrid formed is known to occur in nature to form a stable,
fertile hybrid allotetraploid, A. suecica. This facilitates comparison of established wild allotetraploids to synthetic
laboratory populations. A diagram showing the chromosomal genetics of this model system is shown in Fig. 1.
Early studies revealed that formation of hybrids between
tetraploid A. thaliana and A. arenosa in the laboratory
yielded F1 hybrids with a range of phenotypes, not necessarily intermediate between the parents. A striking observation was that some of the altered phenotypes—such as
meristematic fasciation and pigmentation—were unstable
in hybrid individuals, indicating that dynamic epigenetic
changes affecting gene expression had taken place. Gene
expression analysis using the AFLP-cDNA method to sample expression profiles from both parental genomes has
revealed that approx. 11 % of a 2430 cDNA fragment sample showed changes in expression in hybrid populations
relative to the parents (Wang et al., 2004). This confirms
earlier studies where both gene silencing and gene activation were observed, from both parental genomes (Comai
et al., 2000). To confirm that the gene expression changes
are due to epigenetic differences from the parents, and could
not be attributed to the DNA sequence, the cDNAs displaying these changes have been further analysed.
The first and most compelling question answered by
these studies was: what do these arbitrarily identified
cDNAs encode? As expected, a range of genes were found
even in the first small samples analysed by Comai et al.
(2000), from transposon-related sequences probably
derived from a heterochromatic region to a coding gene
associated with repetitive DNA, to coding genes with no
apparent connection to such sequences. Determination of
DNA methylation of these sequences in the hybrids showed
that they exhibited major differences to their parental
18
Grant-Downton and Dickinson — Epigenetics, Evolution and Beyond
Arabidopsis arenosa
Arabidopsis thaliana
(sporophyte)
(sporophyte)
2n = 4x = 32 (AAAA)
2n = 2x = 10 (TT)
Treatment with
Normal meiotic
colchicine
program
Arabidopsis thaliana
Arabidopsis arenosa
(sporophyte)
( gamete)
2n = 4x = 20 (TTTT)
n = 2x = 16 (AA)
Normal meiotic
program
Arabidopsis thaliana
Pollination, fertilization
( gamete)
and embryogenesis
n = 2x = 10 (TT)
Arabidopsis suecica (TTAA)
2n = 4x = 26
F I G . 1. Formation of synthetic Arabidopsis suecica allotetraploids (T =
A. thaliana genome, A = A. arenosa genome).
counterparts. Later work with a broader sampling has shown
that many coding genes with different functions, and many
with unknown functions, show differential expression in
newly synthesized hybrids (Wang et al., 2004). Interestingly, some of the cDNAs isolated have not shown simple,
stable deactivation or reactivation patterns after hybrid
formation; some have shown patterns of reactivation and
deactivation across selfed generations of the newly synthesized hybrid (Comai et al., 2000; Wang et al., 2004). This is
corroborated by work by Madlung et al. (2002) where
methylation-sensitive amplified polymorphism (MSAP),
assessing cytosine methylation at specific restriction sites
throughout the genome, showed variation in methylation
patterns in selfed hybrid populations. Are the same patterns
of gene expression changes, including this instability, also
found in established natural populations of A. suecica? This
has been addressed by Lee and Chen (2001). By using the
same AFLP-cDNA technique, it was shown that natural
hybrids show differential patterns of gene expression compared with their parental species. Later data from Wang et al.
(2004) has shown, as expected, that there is an overlap in the
gene sets differentially expressed in lines of newly synthesized A. suecica and natural A. suecica. However, these
patterns of gene expression are probably much more stable
in natural A. suecica than in the synthetic laboratory populations, as the wild plants do not exhibit phenotypic instabilities; however, these instabilities return when the plants
are treated with the cytosine demethylating agent
azacytidine (Madlung et al., 2002) or when levels of
DDM1 and MET1 are reduced by transgenic RNAi techniques (Wang et al., 2004). Both these treatments cause
reactivation of gene expression at some of the silent loci
examined.
Models and mechanisms for epigenetic change in hybrids
There is no doubt that this molecular work is hugely
impressive but many questions still remain. The most
disappointing shortcoming has been the inability to connect
an epigenetically derived gene expression change with the
generation of a phenotypic alteration in the hybrid. This will
certainly be challenging in hybrid systems which are not so
easy to work with, but essential to confirm the importance
of epigenetics. Such work would also give an indication of
whether epigenetic changes induced by hybridization could
be of immediate survival or adaptive value. Our opinion is
that both questions will be answered in the affirmative; the
current estimates of the proportion of genes in parental
genomes affected by hybridization, even if too high, and
the fact that coding genes are highly represented supports
our view.
Two further fundamental questions also spring to mind.
First, is this epigenetic instability in hybrids taxonomically
widespread in plants? The answer is almost certainly ‘yes’.
Complex gene expression changes (even more subtle than
those found in the Arabidopsis hybrids) have been found in
the homeologous gene pairs of 40 protein-coding genes in
Gossypium allopolyploid hybrids (Adams et al., 2003),
although confirmation that these are epigenetically derived
is still lacking. However, in a large sample of allopolyploid
hybrids involving Triticum and Aegilops species, Shaked
et al. (2001) have shown that methylation changes were
widespread using AFLP, MSAP and Southern blotting techniques. In one newly synthesized wheat allotetraploid, gene
silencing and methylation changes at multiple loci were
observed (Kashkush et al., 2002). A rough estimate of 1–
5 % of genes silenced by allopolyploid hybrid formation
has been put forward following this work. Evidently monocotyledons also exhibit epigenetic instability in hybrid
formation. The other essential question is: what mechanism
generates epigenetic instability in hybrids? There is no evidence that the affected genes in A. thaliana are clustered as
they map to different locations on the five chromosomes
(Wang et al., 2004). Clearly, some kind of genome-wide
effect is taking place that affects the epigenetic stability
of various loci, in some cases over multiple generations.
Comai et al. (2003) have put forward a model to explain
this phenomenon. Their hypothesis is that during evolutionary time, as two populations diverge and develop into
different species, there is a slight divergence in the protein
components of chromatin regulatory complexes. The subunits of multi-protein complexes that interact with each
other would evolve co-ordinately. When the two species
then form a hybrid, the alteration in the structure of these
complexes through the combination of protein products
encoded by different parental genomes may impair their
function to an extent. This impairment of function in a
hybrid could generate genome-wide alterations in chromatin structure, with changes in DNA methylation as a
secondary effect. A second component of this hypothesis
is that the changes in chromatin regulation allow derepression of silencing of transposable elements, with the hybridization event inducing a burst of transposition. Re-silencing
of these sequences via RNA-based RdDM systems will take
place; however, if a homologous sequence is found ‘naturalized’ in a functionally important region such as in a
promoter or enhancer, this could also lead to coding gene
expression changes. The finding that genes similar to
transposable elements and coding genes associated with
Grant-Downton and Dickinson — Epigenetics, Evolution and Beyond
Formation of normal
transcripts
19
Formation of normal
transcripts
Species A
Species B
HYBRID
FORMATION
Formation of chimeric
transcription factor complex in 1
hybrids with novel sequence
specificity for another site in the
hybrid genome
2
Formation of
aberrant transcripts
Weak transcription from ectopic promoter at new
site
Transcription factor complex from species B
binds to ectopic promoter site in the genome
of species A in the hybrid
Formation of
aberrant transcripts
Weak transcription from ectopic promoter at new
site
F I G . 2. How hybrid formation may lead to the generation of ectopic promoters and aberrant transcripts.
repetitive DNA show altered expression in hybrids is fitting
with this explanation. However, Wang et al. (2004) found
that repetitive/transposable element sequences were not
abundant in the samples of differentially expressed
genes. Perhaps this reflects the efficiency of silencing systems, though if the epigenetic systems of hybrids are to an
extent deregulated this is rather surprising. In the future, it
may be desirable to clone siRNA populations from hybrid
and parental genomes to provide more supportive evidence.
In our view, this model may not be a comprehensive
explanation of gene expression changes for several reasons.
For example, it seems rather incongruous that genes such as
FWA, SUPERMAN and BAL/CPR1 that show great sensitivity to perturbation of components of the epigenetic regulatory system, e.g. ddm1 mutants, have yet to appear
amongst the lists of genes with altered expression in
hybrids. The model of Comai et al. (2003) also does not
sit comfortably with data from studies of differences
between species in components of the epigenetic system.
CenH3, the centromere-specific H3 variant, has been cloned
from both A. arenosa and A. thaliana and, although the
sequences show divergence in the amino terminal region,
whether the amino acid changes have functional relevance
is unknown. The CenH3 of the A. thaliana genome, which
was immunologically distinguishable from the A. arenosa
CenH3, was capable of binding to centromeres of both
genomes. As mitotic and meiotic divisions are not greatly
compromised in A. suecica, this suggests that these two
homeologues are interchangeable in function. More
remarkably, in oat–maize addition lines with stable maize
chromosomes, only the oat CenH3 is incorporated at maize
centromeres (Jin et al., 2004).
Other explanations for reactivation of previously silenced
transposable elements seem possible. It has been shown in
Nicotiana that some transposable elements are exquisitely
sensitive to the physiology and biochemistry of the plant
(such as levels of phytohormones); in a hybrid environment,
such elements from the parental genomes may be exposed
to new conditions that elicit their re-activation (reviewed
in Grant-Downton, 2003). Equally, it seems plausible that
coding genes may have altered expression induced through
generation of ectopic promoters in hybrids (GrantDownton, 2003). Ectopic promoters could be generated
through divergence between the different species in
genomic DNA sequences, their epigenetic modifications
and the transcription factors that recognize them (see
Fig. 2). When combined in a single genomic context, initiation of transcripts from ectopic sites may result and these
would form aberrant RNAs recognized by the RNA surveillance system. The downstream result will be DNA methylation and chromatin modifications at these ectopic
promoter sites and any regions of homology in the hybrid
genome. Empirical support for this theory comes from work
by Tran et al. (2005), where analysis of DNA methylation
patterns in the Arabidopsis thaliana genome has revealed
evidence for ectopic promoters. Unexpectedly, Tran et al.
(2005) found clusters of dense CpG methylation within
genes that may have been stably maintained for generations.
The patterns of these CpG clusters within genes suggests
they may be formed after rare ectopic transcription events,
Grant-Downton and Dickinson — Epigenetics, Evolution and Beyond
20
over several generations, and which parental sequence
of the two is silenced is determined stochastically (Wang
et al., 2004).
A
Epigenetics and other hybrid phenomena
Gene 1.1
Gene 1.2
Hybrid 1 × 2
B
Species
1
Species
2
Hybrid
1 × 2
F I G . 3. Hypothetical model showing outcome of divergence in miRNA
expression patterns in two plant species and their hybrid. (A) Two species, 1
and 2, share the same target gene (gene 1.1 and 1.2) that promotes cell
divisions and outgrowth of a tissue (expression domain indicated by lines).
However, the two species differ in the expression pattern of microRNA
genes (microRNA1 and microRNA2, respectively), that both target this
gene (expression domain marked by lines). (B) After miRNA-induced
degradation, the phenotypic outcome on the tissue is seen. The hybrid differs
markedly from both parents as the actions of both miRNA1 and miRNA2
have limited expression to only a small group of cells in the tissue.
resulting in formation of aberrant RNAs that generate
RdDM. Maintenance of CpG sites by MET1 ensures that
the methylation pattern is preserved even in the absence of
inducing transcripts.
Another conceivable source of gene expression changes
in hybrids may come from divergence in miRNAs and their
targets, and divergence in patterns of miRNA expression
and miRNA target gene expression, between different species. A hypothetical outcome is shown in Fig. 3, where the
developmental outcome on the growth of a tissue is shown
in parental species, and in their hybrid.
As discussed above, miRNAs can evolve with some
rapidity from inverted repeats. Of course, miRNAs downregulating different genes from their original targets would
not necessarily produce a heritable epigenetic mark. Both Li
and Chen (2001) and Wang et al. (2004) provide interesting
evidence that certain sequences are inherently susceptible to
expression changes in allopolyploids, indicating that genes
are not stochastically selected but have some inherent
feature predisposing them to the effects. However, some
sequences show an immediate, stable expression change
in the first generation hybrid that depends on their species
of origin, whereas other sequences establish their silencing
Can epigenetics be invoked to explain other phenomena
seen in hybrid plants? Explaining the molecular basis of
cases of ‘hybrid vigour’ or ‘heterosis’ has been taxing and
an epigenetic perspective may prove to be useful and complementary to conventional genetic explanations (see GrantDownton, 2003; Chandler and Stam, 2004; Grant-Downton
and Dickinson, 2004). Even more strange is the evidence for
rapid genomic changes in plants after polyploidization and
hybridization events. Ozkan et al. (2001) and Shaked et al.
(2001) have presented extensive evidence for elimination of
DNA sequences from a large number of hybrids involving
Triticum, Aegilops and Secale, both diploid and allopolyploid. Elimination of DNA sequences was permanent and
no chimeric tissues were detected, but more strange still was
the discovery that the sequence elimination was consistent,
with the same elimination pattern for a sequence seen in all
the individuals in the same generation of the same hybrid
combinations. These patterns were unchanged even when
the crosses were duplicated with parents of different accession. Eliminated sequences represented not only high-copy
sequences but also low-copy sequences. Another bizarre
twist from the work by Shaked et al. (2001) was the demonstration that a bias in the amount of sequence elimination
from the different parental genomes can occur in some
crosses. Perhaps most significant of all, this work did
show that a substantial number of DNA sequences from
both parental genomes could be eliminated in just a few
generations after a hybridization event. Kaskush et al.
(2002) analysed transcriptome changes in hybrids and,
although it was not possible to provide an accurate quantitative estimate attributable to elimination, these authors
assert that this elimination process could be responsible for
a significant amount of transcript loss. However, this work
did show the identity of one of the eliminated sequences as a
coding gene, Acc-2 (acetyl-coenzyme A carboxylase). The
exact mechanism that induces these elimination patterns
remains unknown. It seems possible that these genomic
changes could be the result of transposon activation in
hybrids, resulting in translocations and rearrangements;
less dramatic genomic changes of a similar kind occur in
Arabidopsis suecica by this mechanism (Madlung et al.,
2005). The consistency of sequence elimination in these
grasses still remains difficult to explain by this mechanism.
Other explanations such as more co-ordinated programmed
genomic rearrangements as seen in the programmed DNA
excision in ciliates, a mechanism that depends on the RNAbased pathways (reviewed in Matzke and Birchler, 2005)
should also be considered.
More dramatic elimination processes are also seen in
some hybrid plants. Uniparental loss of chromosomes is
frequently seen in wide hybrids of grasses (reviewed in
Bennett, 1995), and has also been recorded from other
plants (e.g. Solanum; Clulow et al., 1991). The elimination
processes typically eliminates all the chromosomes of one
Grant-Downton and Dickinson — Epigenetics, Evolution and Beyond
parent during mitotic divisions early in somatic development of the hybrid, though examples where elimination is
incomplete (such as oat–maize hybrids; Riera-Lizarazu
et al., 1996) or biparental are also known. Cytological studies have revealed that the position of chromosomes within
the nucleus is strongly correlated with their retention or
elimination. However, nothing is known about the molecular basis of the elimination process. In some remarkable
cases it has been associated with somatic recombination
between
eliminated
and
retained
chromosomes
(Wilkinson et al., 1995; Pasakinskine et al., 1997). Epigenetic dysfunction at centromeres of eliminated chromosomes in hybrids, affecting the stability of chromosomes
on the mitotic spindle, appears to be the most plausible
explanation (Grant-Downton, 2003). Position effects of
chromosomes in the nucleus may also be responsible for
‘uniparental dominance’ effects in hybrids (Bennett, 1988;
Heslop-Harrison, 1990). Fl hybrids between Hordeum
vulgare and Secale africanum show this phenomenon, as
most morphological and physiological characteristics
resemble the Secale parent. It has been suggested that
extreme distortion in the Hordeum · Secale hybrid arises
from the positional effects of the two different genomes in
the nucleus, with cytological studies showing that the Secale
chromosomes are always displaced peripherally relative to
the Hordeum chromosomes (Heslop-Harrison, 1990). Again,
the molecular basis is unknown and even a transcriptomic
study of these hybrids is needed. It seems possible that
plants also have distinct nuclear territories associated
with transcriptional activity or inactivity, akin to those in
mammalian nuclei (reviewed in Zink and Cremer, 1998;
William, 2003).
EPIGENETICS OF PLOIDY CHANGES
The observant reader may notice that the hybrid examples
discussed above, where epigenetic changes and gene
expression changes have been confirmed, are all allopolyploids. Whether this reflects a genuine difference between
diploid and allopolyploid hybrids, or whether it represents
an experimental bias in current research trends and model
organisms, cannot be determined from the paucity of published papers. There is no doubt that some useful diploid
hybrid systems exist that could be readily examined using
the same kinds of techniques. For example, the hybrids
between Arabidopsis thaliana and A. lyrata would make
a fascinating subject for comparative studies. Equally, the
diploid hybrids in the genus Helianthus, where hybrid
formation has been shown to have the potential to lead to
very rapid speciation by formation of ecophysiologically
and morphologically distinct populations, have been extensively examined at the molecular level by Rieseberg and coworkers (e.g. Rieseberg et al., 1995, 1996, 2003; Rieseberg,
2000; Lexer et al., 2003). Here, the architecture of hybrid
traits was explored by employing DNA sequence markers
and QTL methods, but as these do not have the capacity to
resolve gene expression changes and epigenetic differences
in the same way as the techniques employed in the studies
above, the relative contribution of epigenetic changes to
diploid hybrid speciation is unknown. It would be exciting
21
to integrate data on epigenetics with the fine molecular
mapping and QTL studies employed before, as the relative
contribution of epigenetic variation and sequence variation
to the ‘transgressive segregation’ phenomena of hybridization in Helianthus could be dissected. Indeed, our awareness
of work in epigenetics begs the question whether some
QTL are in fact epialleles, and also highlights the importance of always distinguishing between enzymes that
are methylation-sensitive and methylation-insensitive in
molecular marker studies.
In the work on allopolyploid hybrids, controls were
employed to ensure that gene expression changes were not
derived from the change in ploidy alone, in the case of
Arabidopsis suecica the autotetraploid A. thaliana parent.
Ploidy changes eliciting stable epigenetic changes and gene
expression alterations from transgene loci have been
recorded on a couple of occasions in A. thaliana
(Mittelsten Scheid et al., 1996, 2002). However, Wang
et al. (2004) have shown that even endogenous genes can
be dramatically affected by ploidy change. Genes encoding
a DNA binding protein (DBP) and a putative protein (PP1)
were silenced in newly synthesized autotetraploids, but
when allopolyploid hybrids were formed from them these
genes were reactivated. This suggests that at an epigenetic
level some genes are differentially sensitive to a simple
ploidy change and an allopolyploidy event. This begs the
question of what is the trigger for silencing in autotetraploids, and what allows their subsequent reactivation
in an allopolyploid background. There is also evidence of
sensitivity of maize gene expression to changes in chromosome number giving rise to aneuploidy. As changes in
ploidy in flowering plants appears to be quite a frequent
and easily achieved event, such gene expression changes
may have some evolutionary importance. However, in
Arabidopsis the total number of genes affected in a measurable way appears to be rather small.
Changes in ploidy also affect the ability of many plant
species to form viable hybrids as the endosperm appears
to be particularly sensitive to ploidy balance. For example,
maize hybrids between diploid and autotetraploid maize
result in degenerate endosperm, and seed development is
usually blocked at an early stage. Indeed, even in formation
of allopolyploids where there is greater ploidy balance in
the endosperm, there can be failure in early development of
this nature. For example, in the formation of A. suecica a
large number of F1 embryos do not develop or the seeds
do not show viability, probably in part due to defective
endosperm development (Comai et al., 2000; Bushell
et al., 2003). In many cases of failure of interploidy or
interspecific crosses, the embryo can be rescued and developed in vitro. In both situations, the cause of defective
development is most likely rooted in aberrant expression
of imprinted genes necessary for endosperm development.
In the case of interploidy crosses, the effect of imprinted
genes is simply quantitative, the outcome of endosperm
development in the cross dependent on copy number of
the genes (with ploidy level directly determining copy
number) and their parent-of-origin. In interspecific crosses,
the effects are much more complex; differences between
species (even of the same chromosome number) may exist
22
Grant-Downton and Dickinson — Epigenetics, Evolution and Beyond
F I G . 4. Epigenetic control of floral symmetry in Linaria vulgaris (common toadflax). Right: wild-type L. vulgaris where the Lcycloidea locus controlling
dorso-ventral patterning is expressed normally. Note the zygomorphic flower with the ventral petal lobe displaying a spur. Left: the peloric epimutant shows
hypermethylation of the Lcyc sequence and loss of expression of Lcyc. The flowers show greater radial symmetry as the ventral petal lobe is repeated five
times. The plant shown here is homozygous for the Lcyc epiallele; heterozygotes show normal floral development.
in copy number and sequence/function of imprinted loci,
and even presence or absence of some imprinted genes,
will affect endosperm development, in addition to parentof-origin effects. Overlaid on this may be further complexities in gene expression patterns as discovered in the 2n
sporophytic tissues. Epigenetic misregulation in endosperm
at imprinted loci, leading to abortion of the developing
seed, must be a powerful isolating mechanism in plant
speciation events. As it can be set up by a simple ploidy
change, it could be a hugely effective mechanism to achieve
reproductive isolation and initiate speciation events. Even
changes in copy number or expression levels of one or two
imprinted loci have the potential to set up strong reproductive isolation barriers between different populations.
Equally, as allopolyploidy events show, a ploidy change
can bring down barriers between species. It is important
to note that some plants do not have this reproductive
barrier, e.g. hybrids between Papaver somniferum and different species of Papaver in Section Oxytona which show
different ploidies, as well as hybrids within section Oxytona,
form normal, viable seeds (Ojala and Rousi, 1986; Ojala
et al., 1990). Furthermore, it seems more than a coincidence
that some plant families which readily form ‘wide’ hybrids
(such as intergeneric hybrids) also show a highly pared
down endosperm—the Orchidaceae and Gesneriaceae, for
example.
THE INTERFACE BETWEEN EPIGENETICS
AND OTHER FIELDS
As can be seen from the previous section, epigenetics and
the evolution of plants is inextricable. Yet despite this now
obvious statement, epigenetics is seldom discussed in a
context of taxonomy and systematics, or population biology
and conservation. Only one recent review, by Kalisz and
Purugganan (2004), has considered the importance of
epialleles in population genetics and evolution. Here, an
attempt is made to redress the balance.
Taxonomy and systematics
The first encounter between epigenetics and systematics
occurred at the birth of modern systematics when Linnaeus
became aware of the ‘monstrous’ peloric variant of Linaria
vulgaris, now known to be caused by hypermethylation of
Lcyc. So different in basic floral morphology was this spontaneous variant, illustrated in Fig. 4, it caused Linnaeus
considerable concern (for historical review, see
Gustaffson, 1979). The fact that epigenetic variants can
generate stable morphological changes in plants, to the
point of homeotic transformations, cannot be ignored any
longer by those studying plant systematics. As much taxonomy is still based on analysis of morphological features, it
would seem that an obvious deduction is that some plant
species may have been misclassified, and merely represent
epigenetic variants of one species. This problem may be
particularly acute in taxa where hybridization is frequent
and from locations where sampling is patchy. Conversely,
by virtue of the ease of reproductive isolation through
ploidy change and, perhaps, through alterations in imprinted
genes without associated ploidy changes, there may be more
‘cryptic’ species in plants, difficult or impossible to distinguish by morphology, than previously assumed.
Another consideration dictated by the epigenetic revolution relates to the evolution of plant characteristics. If, in a
given taxonomic group, some genes controlling certain
characteristics (e.g. stamen number, floral symmetry) are
initially (or become) unusually labile at the epigenetic level,
it would be expected that these characteristics would be
more subject to repeated modification, loss or gain in the
clade. In phylogenies of certain taxa, the frequent change or
reversibility of some characteristics could be rooted in
Grant-Downton and Dickinson — Epigenetics, Evolution and Beyond
epigenetics. This idea will be illustrated by two different
examples. A gene susceptible to stable silencing by hypermethylation of the promoter may become furtherentrenched
in an inactive state by virtue of increased rate of mutation of
the methyl-cytosine leading to functional change. Alternatively, when a gene is regulated by a miRNA, just a few
nucleotide changes in the site of complementary binding to
the miRNA could render the mRNA less susceptible or
completely immune to regulation. Ectopic or over-expression by this mechanism could result in modification, loss or
gain of a characteristic.
We have already hypothesized that rare saltatory changes
in multiple characteristics could come about by virtue of
aberrations in the epigenetic machinery. To reiterate, there
is also supportive evidence that allopolyploid hybridization
can lead to widespread epigenetic changes, altered gene
expression and phenotypic changes, so allopolyploid speciation can be rapid and involve changes in multiple characteristics. More fascinating still is the (frustratingly
incomplete) evidence for substantial differences in epigenetic systems in the gametophyte between different
angiosperm taxa. Even if such a saltatory event occurs
very infrequently in a phylogeny, its manifestation may
create an interesting situation. Traditional morphologybased analysis may face considerable problems in resolving
the taxonomy and phylogeny of a clade where an event of
this kind has happened. The occurrence of such events may
assist in the explanation of why phylogenies based on the
DNA sequence sometimes show that morphological change
is out of phase in relation to change to the DNA sequence.
Epigenetics may illuminate and increase the complexity of
the debate on molecules versus morphology in plant
systematics.
Population genetics and conservation
Implicit in the discussion above is that epigenetic regulation and epigenetic variation has the capacity to generate
phenotypic variation that may (or may not) have adaptive
value. Unfortunately, measuring epigenetic variation in
plant genomes is much more complex than determining
variation in DNA sequences. For example, digestion of
genomic DNA with isoschizomers can reveal DNA methylation patterns, whilst bisulfite sequencing can reveal in
detail the methylation patterns of individual sequences.
However, unlike the relatively static DNA sequence, methylation patterns can vary dramatically upon even the same
DNA sequence, depending on factors such as environment
and developmental stage, making sampling more complex.
For instance, genomic DNA methylation levels have been
shown to increase with developmental age, from seedling to
mature plant, even in ephemeral species such as Arabidopsis
thaliana (Ruiz-Garcı́a et al., 2005). This adds another new
set of problems to generating data on DNA methylation
patterns. At least there are some data for variation in
populations of A. thaliana, with considerable variation
between ecotypes in methylation detected at rRNA gene
repeats (Riddle and Richards, 2002). Detectable differences
between ecotypes at the sequence level of the rRNA genes
were negligible, though substantial differences in gene copy
23
number were evident. QTL analysis of the control of this
trait was performed by Riddle and Richards; unsurprisingly,
the major determinant of methylation mapped to the rRNA
repeats themselves is probably due to the strong inheritance
of parental epigenetic states at these loci. However, this
could not account for all of the variation between ecotypes
and trans-acting modifier loci affecting methylation
whichwere shown to exist. These QTL loci contained strong
candidates for genes controlling methylation such as KRYPTONITE and DNA methyltransferases. Of course, DNA
methylation patterns are but one facet of epigenetic variation. The technology to assess variation in histones on a
sequence-by-sequence basis and siRNA populations is in its
infancy.
Measuring sequence variation by methods such as RAPD,
RFLP, AFLP and VNTR amplification (for reviews, see
Karp et al., 1996; Mueller and Wolfenbarger, 1999) will
only give an estimate of variation in the DNA sequence.
How much epigenetic variation is stored by the genomes of
a population cannot be adequately described by present
techniques, and a more detailed picture of the epigenetic
variation in a model species, say Arabidopsis, would be a
major step forward. How important is this otherwise ‘cryptic’ variation? Epigenetic data superimposed upon sequence
data is likely to greatly improve the association of markers
and traits, showing both continuous and discontinuous
variation, in studies of populations. Another impact is
that many repetitive sequences in genomes, previously considered neutral or nearly neutral, may emerge to show
effects on traits and fitness. By affecting transcription of
key genes, repetitive sequences and the chromatin they form
may impact on quantitative variation and even effects on
fitness. For example, some diseases in humans are now
known to map to an increase in size of a repetitive DNA
(reviewed in Sinden et al., 2002) and the structural effects
generated by repeat expansion may block transcription of
the gene (Sakamoto et al., 1999, 2001). These effects may
be local to exceptionally non-local. A superb example of the
latter is paramutation at the B locus in maize, where the
repetitive region, a tandem array, exhibiting the epigenetic
changes that are governing paramutation behaviour is
100 kb upstream of the b1 coding region (Stam et al.,
2002). Even by smaller influences on factors such as chromatin structure that affect chromosomal architecture and
recombination events, and the distance between enhancers
and coding sequence, repetitive DNA can have a nonneutral and non-local effect on other sequences. With repetitive sequences emerging time and time again as a key target
of epigenetic modification, selection must always be acting
on repetitive sequences and it is quite wrong to dismiss these
sequences as ‘junk’ evolving out of tempo with coding
sequences.
What happens to epigenetic variation in populations
affected by artificial pressures exerted by humans, more
precisely in wild populations subjected to disruption by
man and those in cultivation exposed to artificial selection
(plant domestication and plant breeding)? Conservation
biologists interested in conserving variation in plant populations should now heed the fact that epigenetic variation
may be significant but cannot be readily measured.
24
Grant-Downton and Dickinson — Epigenetics, Evolution and Beyond
For example, in plant populations, epigenetics has been
shown again and again to have a tremendous influence
on plant fertility. In disrupted environments or through
exposure to over-exploitation, small or fragmented wild
populations may be subjected to in-breeding; fixation of
mutations in the epigenetic machinery that reduce fertility
(amongst other effects) may have an impact on the longterm population genetics and even survival of the sub-populations. On the other hand, epigenetic variation induced in
such backgrounds may have the opposite effect, allowing
survival of the population in the face of abiotic and biotic
adversity such as higher grazing pressure and climate
change, by altering traits of selective value such as plant
stature and flowering time. Metastable alleles at some loci
may allow temporary escape of unfavourable conditions
with considerable rapidity and without the permanence of
nucleotide changes. Consequently, carrying or generating
an ‘epimutational load’ may have advantages and disadvantages. On the other hand, epigenetic changes may assist in
explaining why hybrids between native and non-native species may be unusually adaptable and become highly invasive. The invasive allopolyploid Spartina anglica, formed
less than 150 years ago between a native and an introduced
species, would be an interesting candidate for investigation.
Surprisingly, genetic analysis of wild S. anglica clones
showed no significant genetic changes compared with the
parental species, even showing relative quiescence of transposable elements (Baumel et al., 2001, 2002). Recently,
significant epigenetic changes have been revealed using
MSAP analysis (Ainouche et al., 2003). In the invasive
Senecio hybrids formed between S. squalidus (2n) and
S. vulgaris (4n), the infertile S. baxteri (3n) and the derived
fertile S. cambrensis (6n), there are significant changes to
the transcriptome in the hybrids compared with the parental
species (Hegarty et al., 2005). Unfortunately, these have yet
to be associated with epigenetic changes.
The pool of epigenetic variation, and the comparative
ease with which it can be modified, may help to explain
why some naturalized species can establish and become
invasive in a short time, even though the naturalization
process is accompanied by a massive bottleneck in genetic
variation. An epigenetic perspective may also be useful in
understanding how natural population bottlenecks in founder events can lead to formation of viable colonies and
(eventually) speciation in isolated environments such as
islands. In conservation biology, epigenetics may have
much use, and questions such as whether epigenetic diversity is eroded in a similar way to genetic diversity in plant
populations under pressure should be addressed in the
future.
In plant domestication and plant breeding, population
bottlenecks are also a feature, often together with hybridization and ploidy changes. A notable feature of artificial
selection is that novel or extreme traits are frequently the
object of selection, whether the effort is deliberate or unconscious. Are epigenetic variants selected for under these
conditions? In clonally propagated plants, selection may
particularly favour epigenetic variants as even non-heritable
or metastable epigenetic states could be propagated
indefinitely. Efforts are definitely needed to address this
question, as it may help to explain why genetic diversity
can drop dramatically yet phenotypic variation and phenotypic plasticity remains high. An outstanding example is
analysis of AFLP variation in cultivated hybrids in
Hemerocallis (Tompkins et al., 2001). This genus is
particularly useful as hybridization and selection from the
handful of progenitor species, such as H. citrina and
H. altissima, is only just over a century old but the original
clones of the species remain in cultivation. As an intense
level of selection has proceeded, producing cultivars
increasingly novel and phenotypically divergent from the
progenitor species, genetic diversity has decreased dramatically. This drop in genetic diversity is particularly sharp in
tetraploids, a ploidy state derived artificially by plant breeders using colchicine and similar compounds. It would be
interesting to determine whether epigenetic diversity in
hybrid cultivars compared with progenitor species
has decreased, remained stable or increased during this
selection.
CONCLUSION
In this two-part review, a basic introduction to epigenetics
in plants has been given. It has highlighted that beyond the
intrinsic interest in molecular biology, an epigenetic perspective on plant evolution, systematics, population biology
and conservation is now needed. There is hope that the
‘epigenetic revolution’ will initiate and influence thinking
and experiments in these fields to a much greater extent in
the future.
FOOTNOTE ADDED IN PROOF
During the production of this review, an outstanding and
important paper has emerged which highlights how little is
still known about the interactions between epigenetic and
genetic systems in plants. Homozygous loss-of-function
mutations in the HOTHEAD locus of Arabidopsis were
shown by Lolle et al. (2005) to spontaneously correct the
nucleotide change generating the mutant hothead allele in
question, and consequently revert to wild type. This correction was specific and well beyond the frequency expected
from background mutations. Furthermore, HOTHEAD
mutants showed the same enhanced correction of other
mutations in disparate parts of the genome, both coding
and non-coding regions. Although the mechanism remains
obscure, it is proposed that plants must carry a cache of
replica genomic information, possibly encoded by RNA
species that is passed across generations and subsequently
used as a template. Correction of mutations by these ‘ghosts
of genomes passed’ is held to be effected by some part of
the repair system, and may be useful in adapting to various
stresses such as abiotic stress and in-breeding depression.
How this tantalizing discovery relates to the epigenetic
systems described above, and how it relates to more complex genomic situations in ploidy change and hybridization,
remains a fascinating prospect for future research.
Grant-Downton and Dickinson — Epigenetics, Evolution and Beyond
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